Determination of transgene copy number and expression level using denaturing gradient gel electrophoresis.
Transgenic mice and cell lines are frequently developed to study human disease. Accurate determination of transgene copy number and levels of mRNA are necessary to understand the phenotypic changes observed in these models. Currently, transgene copy number and expression are estimated by Southern blot analysis of genomic DNA and Northern blot analysis of mRNA. We report a novel PCR-based method for determining transgene copy number and levels of transgene expression using competitive PCR between endogenous genomic genes and mutant transgenes followed by denaturing gradient gel electrophoresis (DGGE). We are able to accurately quantify a range of 1-10 copies of transgene incorporated per diploid genome. After reverse-transcribing RNA to cDNA, we are able to quantify levels of transgene mRNA that correlate with biochemical and histological evidence of transgene activity. In conclusion, resolving PCR and reverse transcription-PCR products by DGGE is a rapid and reproducible method that allows for accurate determination of transgene copy number and expression. This technique provides a more complete understanding of transgene effects.
Ringel
M D
MD
Johns Hopkins University, School of Medicine.
Schwindinger
W F
WF
Saji
M
M
Zeiger
M A
MA
Levine
M A
MA
D000818:Animals; D015139:Blotting, Southern; D004586:Electrophoresis; D018628:Gene Dosage; D051379:Mice; D008822:Mice, Transgenic; D016133:Polymerase Chain Reaction; D012333:RNA, Messenger / Q000032:analysis; D019076:Transgenes
D012333:RNA, Messenger